Project Summary/Abstract The candidate seeks to acquire additional research and career development training through a plan designed to address deficiencies in her current expertise. The plan will build on her existing skills and knowledge in the study of a new system which she will carry with her into the next phase of her career. She will use the mentored phase of this award to build skills in instrument design, personnel management, and manuscript development and publication. This training will enable her to acquire the additional skills necessary to transition to independence and establish a successful research program. The proposed research will use cutting edge single-molecule and computational techniques to study the enzymatic activity of the DNA-unwinding complex TFIIH. TFIIH opens DNA for both transcription and DNA repair, but utilizes two separate helicase domains with distinct mechanisms to do so. The proposal employs a combination of single-molecule magnetic tweezers studies and molecular dynamics simulations to: characterize the activity of the individual helicase domains of TFIIH (Aim 1); investigate the impact of the two helicases on each other in the full TFIIH complex (Aim 2); and explore the differences in DNA preference of the two helicases and the aspects of DNA structure that lead to dominance of one over the other (Aim 3). Completion of this research project will contribute to the fields of both transcription and nucleotide excision repair by elucidating the ways in which TFIIH links these two pathways. The results of the proposed research will provide a foundation for further studies of TFIIH activity and the factors that influence it, and form the basis of the candidate's first R01 proposal. The K22 award will provide the candidate with the opportunity to develop new research skills through additional training during the mentored phase, which will be funded by the NHLBI Division of Intramural Research. These skills include construction of a combined magnetic tweezers/single molecule fluorescence microscope, strategies for the design of DNA substrates for single-molecule experiments, and data analysis methods. During the mentored phase of the award she will also attend courses and seminars designed to impart management and teaching skills and supervise a post-baccalaureate trainee. She will attend workshops on manuscript preparation and build on her current publication record. The training plan also includes career development activities such as practice job talks, mock interviews, and grant writing workshops. These activities will provide her with invaluable experience and skills that she will draw on as an independent investigator.